共表达ETEC 987P、K88重组乳杆菌构建及其免疫原性分析  

Construction and Immunogenicity Analysis of Lactic Acid Bacteria Vector Vaccine with Recombinant Escherichia coli 987P and K88 Proteins Demonstrated on the Surface

作　　者：杨雨晴 李树东[1] 孙晓莹 陈小燕 白永飞 李颖 蔡昌福 余丽芸[2] 侯喜林[1] Yang Yuqing;Li Shudong;Sun Xiaoying;Chen Xiaoyan;Bai Yongfei;Li Ying;Cai Changfu;Yu Liyun;Hou Xinlin(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319;College of Life Science and Technology,Heilongjiang Bayi Agricultural University)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319 [2]黑龙江八一农垦大学生命科学技术学院

出　　处：《黑龙江八一农垦大学学报》2022年第2期44-51,106,共9页journal of heilongjiang bayi agricultural university

基　　金：省高校产业化前期培育项目(仔猪大肠杆菌腹泻病黏膜疫苗开发:1253CGZH14);研究生创新项目(截短的ETEC 987P、K88二联重组干酪乳杆菌的构建及免疫原性分析:YJSCX2019-Y29)。

摘　　要：构建表达产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)987P和K88蛋白的重组干酪乳杆菌,并对重组干酪乳杆菌免疫原性进行分析。通过PCR构建重组质粒pLA-987P-K88并电转化至干酪乳杆菌6105后进行表达,通过Western blot、间接免疫荧光和流式细胞术检测目的蛋白的表达情况。将重组干酪乳杆菌灌胃免疫BALB/c小鼠,三次免疫后用ELISA检测小鼠血清中IgG抗体和肠冲洗液中sIgA抗体水平,采用CCK-8检测淋巴细胞的增殖情况,用C83916(987P)株和C83902(K88)株进行攻毒保护性试验检测其保护率。结果显示,重组干酪乳杆菌成功表达987P-K88融合蛋白,小鼠免疫重组菌后血清中IgG抗体水平和肠冲洗液sIgA水平显著升高,细胞免疫水平明显增长,攻毒保护性试验结果显示,重组菌能同时对C83916(987P)株和C83902(K88)株具有良好的保护作用。The recombinant Lactobacillus casei(L.casei)expressing Enterotoxigenic Escherichia coli(ETEC)987P and K88 prote-ins was constructed,and the immunogenicity of the recombinant L.casei was analyzed.The recombinant pLA-987P-K88/E.coli was constructed.The positive plasmid pLA-987P-K88 was transformed into L.casei 6105 for expression.The fusion target protein was detected by Western blot,indirect immunofluorescence and flow cytometry.BALB/c mice were intragastrically immunized with pLA-987P-K88/L.casei.After immunization for three times,the serum IgG level of mice was detected by ELISA,lymphocyte proliferation was detected by CCK-8,and the protection rate was detected by challenge protection test with C83916(987P)strains and C83902(K88)strains.The recombinant Lactobacillus casei successfully expressed 987P-K88 fusion protein.After immunization with the recombinant Lactobacillus casei,the level of IgG antibody in serum and the level of sIgA in intestinal lavage fluid were significantly increased,suggesting the mucosal immunity was significantly strengthen.The results of the challenge test showed that the recombinant L.casei had of the protective effect against ETEC strains C83916(987P)and C83902(K88)at the same time.

关 键 词：产肠毒素大肠杆菌 987P K88 重组干酪乳杆菌 免疫原性 

分 类 号：S581[农业科学—作物学]